PROJECT SUMMARY BRD4, a major BET (bromo and extra terminal) family transcription regulator, plays a pivotal role in ordered gene transcription in chromatin through its characteristic tandem acetyl-lysine binding bromodomains (BrDs). BRD4 is widely recognized as a promising anticancer drug target from studies using BET BrD inhibitors, some of which are being evaluated in human clinical trials for cancer. However, BET inhibitors are mostly effective in hematopoietic cancers, but much less so in solid tumors including breast cancers. The opening question is why chemical inhibition of this general transcription regulator affects only a limited number of genes and is highly sensitive to cell- and tumor-types. Our recent studies suggest that the mechanism by which BRD4 regulates transcription in chromatin is likely far more complex than the current simplistic view of BrDs binding to acetylated histones and transcription proteins and influenced by context- dependent coordinated activities of its tandem BrDs. We show that a conformationally optimized bivalent BET BrD inhibitor that simultaneously inhibits the tandem BrDs of BRD4 affords sustained repression of BRD4 transcriptional activity by blocking its association with enhancer/ mediator proteins with potency far superior to monovalent BET inhibitors, resulting in inhibition of proliferation of solid tumor cells including a panel of triple negative breast cancer (TNBC) cells and even JQ1 resistant TNBC cells. Our study provides direct experimental evidence on the cell-type and context dependent BRD4 functions in cancers and suggests a new therapeutic strategy to maximally control BRD4 activity required for rapid solid tumor cell proliferation such as the devastating TNBC that currently lacks targeted therapy. Motivated by our promising new findings, in this project, we will develop next-generation BRD4-selective bivalent BrD inhibitors and characterize the transcriptional mechanism and therapeutic potential of BRD4 as a new targeted treatment for TNBC.